Yersinia pestis is the causative agent of plague, a natural disease of mice, rats, and other rodents which also affects man. two other species in the same genus, Yersinia pseudotuberculosis and Yersinia enterocolitica, also cause human disease. Each of these invasive pathogens carries a plasmid approximately 75 kilobases n size which is essential to pathogenesis: either loss of the plasmid or appropriate plasmid mutations results in complete loss of virulence. The 75 kilobase plasmids of all three species are closely related. Two important features apparent in all of these species are (1) complex regulation of plasmid virulence genes by temperature and Ca 2+ concentration and (2) plasmid-dependent development of resistance to phagocytosis. Regulation by temperature and Ca 2+ is believed to control expression of virulence genes in three important environments encountered by the bacteria: outside a mammalian host (low temperature), inside a host, but outside other the hosts cells (high temperature, high Ca 2+) and within host cells, such as machrophages, in which these bacteria can grow (high temperature, low Ca 2+). Resistance to phagocytosis is controlled by this regulatory system. It is thought to develop early in infection, perhaps as the bacteria are sheltered within macrophages, and act to protect extracellular bacteria from attack by polymorphonuclear leukocytes during later stages of disease. this activity is the plasmid- determined property most logically related to pathogenesis and may well account for the critical role of he 75kb plasmids in virulence. The mechanisms of both regulation and resistance to phagocytosis are poorly understood. The long-term goals of this project are to determine the molecular mechanisms of both resistance to phagocytosis and the regulatory system which controls it, and determine their role in pathogenesis. This will be accomplished through genetic and biochemical analysis of the genes and gene products of the 75 kb plasmid of Y. pestis, and by analysis of the interaction of both mice and phagocytic cells with Y. pestis strains bearing well-defined mutant plasmids. Y. pestis carries plasmids other than the 75 kilobase species which are not found in the other Yersinia and may account, at least in part, for the systemic and fulminant character of plague. From the 9.5 kilobase plasmid commonly found in Y. pestis strains, we have recently defined and sequenced a gene, designated pla, which encodes both plasminogen activator and coagulase activities. We think it likely that products of this gene are important in the ability of Y. pestis to disseminate rapidly from a periferral site of infection and in two important features of plague pathology: severe disseminated intravascular coagulation and hemorrhaging. As part of this project we will test these hypotheses by analyzing the effect of pla mutations on the coarse of disease and the resulting pathology in mouse infection experiments. These studies will provide information fundamental to understanding the function and regulation of virulence genes in the yersiniae, and should also prove useful int he design of improved plague vaccines.